Developing intranasal dsRNA molecules as broadly effective therapeutics against highly pathogenic coronaviruses

Long double-stranded (ds) RNA molecules are produced by viruses during their replicative cycle. These dsRNA molecules are long (1000s of base pairs) and contain viral genome sequence. In vertebrates, it was thought that viral dsRNA's major role was to stimulate host antiviral type I interferons (IFNs). The IFN response is a broad-spectrum antiviral response, stopping the virus in its tracks. It is potent, but also energetically costly, shuts down the cell's ability to make any protein, and induces side effects such as inflammation. In invertebrates and plants, long dsRNA does not induce IFN, but is used as a substrate for RNA interference (RNAi). RNAi uses an enzyme, Dicer, to cut the viral dsRNA into ~20bp pieces called short interfering (si)RNAs, which are loaded into RISC, where an mRNA whose sequence matches the siRNA is found, bound, and sequestered or degraded. In this RNAi pathway, it is the viral sequences that are targeted for degradation specifically, making it less energetically costly with lower side effects. We have found that long dsRNA can be used as a substrate for RNAi in vertebrate cells if used at a dose that is below the threshold to induce IFNs. Using this strategy, dsRNA containing coronavirus sequences knock down coronavirus (CoV) replication in human cells. The proposed project has 3 aims: (1) identify highly conserved CoV genomic sequences to develop pan-CoV dsRNA sequences that are optimized for Dicer recognition, (2) validate efficacy of dsRNA molecules in inhibiting CoV replication in human airway cells, either 'naked' or encapsulated in a lipid nanoparticle or virus-like particle, and (3) validate the antiviral effects of long dsRNA molecules in the airway using the golden Syrian hamster model of SARS-CoV-2. This project will develop a first-in-its-class pan-coronavirus therapeutic to be used prophylactically to protect the global population from the current SARS-CoV-2 pandemic as well as emerging CoVs.